WO2020166726A1

WO2020166726A1 - Method for preparing quiescent hepatic stellate cell, and model for evaluation of activation of quiescent hepatic stellate cell

Info

Publication number: WO2020166726A1
Application number: PCT/JP2020/006147
Authority: WO
Inventors: 宮島　篤; 丈友木戸; 悠太厚井
Original assignee: 国立大学法人東京大学
Priority date: 2019-02-15
Filing date: 2020-02-17
Publication date: 2020-08-20
Also published as: JPWO2020166726A1; JP2024147691A

Abstract

The present invention addresses the problem of providing: a method for preparing a quiescent hepatic stellate cell by inducing the differentiation of a pluripotent stem cell; and a method for screening for a chemical substance involved in the activation of a quiescent hepatic stellate cell. According to the present invention, a method for preparing a quiescent hepatic stellate cell is provided, which comprises a step of inducing the differentiation of an iPS cell and fractionating a quiescent hepatic stellate cell by employing a NGFR-positive phenotype as an index. Also provided is a screening method for selecting as a candidate substance that can be used as a drug for treating or preventing a fibrosis-related liver disease on the basis of the expression amount of a gene marker that is expressed specifically in a hepatic stellate cell that is activated by introducing a reporter gene into the quiescent hepatic stellate cell.

Description

Preparation method of resting hepatic stellate cells and activation evaluation model

The present invention provides a method for preparing quiescent hepatic stellate cells by inducing differentiation of pluripotent stem cells, quiescent hepatic stellate cells obtained by the preparation method, a cell suspension containing the same, and quiescent hepatic stellate cells. The present invention relates to a screening method for a drug involved in the activation of

Proliferation and accumulation of connective tissue of the liver causes circulatory injury of the liver, which causes hepatic parenchymal cell damage, further proliferation and accumulation of connective tissue, and causes liver disease accompanied by cirrhosis and liver fibrosis. It is considered. The liver consists of parenchymal cells and non-parenchymal cells (hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, Pit cells), and connective tissue of the liver is composed of extracellular matrix (collagen etc.) and cells localized there. Has been done. The hepatic stellate cell (HSC), which is a substrate-producing cell in connective tissue, is activated and transformed to promote the growth and accumulation of connective tissue. The hepatic stellate cells of normal liver (hereinafter referred to as “quiescent hepatic stellate cells”) produce a small amount of extracellular matrix, change their morphology into myofibroblast-like cells upon activation, and increase in number as the number of cells increases. It is known to synthesize the extracellular matrix of.

Therefore, there is a need for a therapeutic method and a prophylactic method for suppressing the activation of hepatic stellate cells in patients with liver diseases associated with liver fibrosis such as liver cirrhosis, and for reducing the growth and accumulation of connective tissue. It can be said that screening for drugs to be suppressed is important. Despite such a need, an efficient screening method for a drug that suppresses the activation of hepatic stellate cells is not known.

The present inventors have succeeded in establishing a differentiation induction system from pluripotent stem cells to hepatic stellate cells (Patent Document 1; Non-Patent Document 1). However, the induced hepatic stellate cells have been shown to be already activated or to be activated immediately after the culturing, like the commercially available and commercially available primary cultured hepatic stellate cells. It is not a useful tool for research and development of drug discovery such as drugs. Therefore, it is desired to develop a drug screening system that reflects the activation process of hepatic stellate cells in a culture system.

International Publication No. 2016/148216

As described above, commercially available primary cultured stellate cells have already been activated, and it is impossible to obtain a large amount of quiescent hepatic stellate cells sufficient to construct a drug screening system from the human body. Therefore, the present invention aims to establish a differentiation induction system from pluripotent stem cells (eg, iPS cells) to quiescent hepatic stellate cells, and to develop an activation control system thereof.

The present inventors have succeeded in obtaining hepatic stellate cells that stably retain a stationary phase from iPS cells by improving the conventional method for inducing differentiation of iPS cells to obtain hepatic stellate cells. Successfully completed.

That is, the present invention has the following aspects.
[1] A method for inducing differentiation of pluripotent stem cells to prepare quiescent hepatic stellate cells, which comprises a step of three-dimensionally culturing pluripotent stem cells and/or a step of culturing at a low oxygen concentration ..
[2]
The preparation method according to [1] above, wherein pluripotent stem cells are cultured in the presence of about 4% O ₂ from the 0th to 6th day of culture.
[3]
The preparation method according to the above [2], wherein after the 6th day of culture, the culture is performed with the O ₂ concentration being the same as in the atmosphere.
[4]
The preparation method according to any one of the above [1] to [3], wherein the three-dimensional culture is spheroid culture, suspension culture or gel culture.
[5]
The preparation method according to any one of the above [1] to [4], which comprises a step of adding a Rock inhibitor.
[6]
The preparation method according to any one of [1] to [5] above, which further comprises a step of incorporating a reporter gene into pluripotent stem cells before differentiation induction.
[7]
The preparation method according to any one of the above [1] to [5], further comprising a step of incorporating a reporter gene into a quiescent hepatic stellate cell during or after the differentiation induction process.
[8]
A quiescent hepatic stellate cell prepared by the method according to any one of [1] to [7] above.
[9]
A cell suspension containing quiescent hepatic stellate cells according to [8] above.
[10]
A screening method for selecting a test substance that suppresses the activation of hepatic stellate cells as a candidate substance used for a drug for treating or preventing liver disease associated with fibrosis,
(A) culturing a quiescent hepatic stellate cell into which a reporter gene has been prepared, prepared by the method according to [6] or [7] above;
(B) quiescent by adding a drug that activates quiescent hepatic stellate cells to the culture system of quiescent hepatic stellate cells, or collecting and reseeding the quiescent hepatic stellate cells and culturing in two dimensions Activating hepatic stellate cells to obtain activated hepatic stellate cells;
(C) adding a test substance to a culture system of activated hepatic stellate cells;
(D) comparing the expression level of the reporter gene before and after the addition of the test substance; and (e) for treating or preventing liver disease when the expression level of the reporter gene decreases after the addition of the test substance A screening method comprising selecting as a candidate substance used for a drug.
[11]
A kit for use in the screening method described in [10] above, which comprises the quiescent hepatic stellate cells described in [8] above or the cell suspension described in [9] above.

According to the present invention, it is possible to prepare non-activated quiescent hepatic stellate cells from iPS cells. The obtained drug can be used for a system for screening a drug for treating a liver disease associated with liver fibrosis such as cirrhosis and evaluating the therapeutic drug.

FIG. 1 shows a method for inducing differentiation of human iPS cells to prepare quiescent hepatic stellate cells. The photographs in the figure are images of morphological changes in spheroid formation in the process of differentiation induction of human iPS cells. FIG. 2 shows an undifferentiated marker gene (OCT4, NANOG), a mesodermal marker gene (MESP1, T), and a transseptal mesenchyme/hepatic star during the differentiation process of human iPS cells into quiescent hepatic stellate cells. The result of having analyzed the expression with time of a cell marker gene (HLX, LHX2, FOXF1) is shown. FIG. 3 is a diagram showing the effect of inducing differentiation into stationary hepatic stellate cells derived from human iPS cells by the presence or absence of addition of a Rock inhibitor (Y27632) to the culture system. In the figure, NGFR and LRAT are marker genes for quiescent hepatic stellate cells, and ACTA2 and COL1A1 are marker genes for activated hepatic stellate cells. FIG. 4 shows an experimental method for examining the oxygen concentration in culture in inducing differentiation of quiescent hepatic stellate cells of human iPS cells and the results thereof. When the oxygen concentration is 4% from the 1st to 6th day after the initiation of differentiation induction, and after the 6th day, the oxygen concentration is maintained at 4% and changed to 20% (corresponding to the atmospheric oxygen concentration). Compared with. A comparative study was performed on the relative expression of the activated hepatic stellate cell marker genes, ACTA2 and COL1A1. FIG. 5 shows the results of examining the gene expression of each group by separating the NGFR+ cells (“posi”) and the NGFR− cells (“nega”) by the MoFLo XDP cell sorter on the 8th day after the initiation of differentiation induction. .. In the figure, “bulk” refers to a cell group before separation by a cell sorter. FIG. 6 shows an image of quiescent hepatic stellate cells obtained by inducing differentiation from iPS cells. FIG. 7 shows each gene in iPS cell-derived hepatic stellate cells (“Ver. 1”) prepared by the conventional method and iPS cell-derived quiescent hepatic stellate cells (“Ver. 2”) prepared by the method of the present invention. The result of having comparatively examined expression is shown. FIG. 8 shows the results of comparative study of gene expression in primary cultured hepatic stellate cells (“hHSC”) and iPS cell-derived resting hepatic stellate cells (“iPS-HSC(Ver2)”) prepared by the method of the present invention. Indicates. FIG. 9 shows the results of examining the vitamin A storage capacity in stationary phase hepatic stellate cells by flow cytometry after adding 10 μM retinol to a culture system of iPS cell-derived stationary phase hepatic stellate cells and culturing for 4 days. FIG. 10 shows the experimental method for activating the stationary hepatic stellate cells derived from iPS cells and the result of the expression of the activated hepatic stellate cell marker genes (ACTA2 and COL1A1). FIG. 11 shows the results of evaluating the activating capacity by inducing differentiation of quiescent hepatic stellate cells from iPS cells. The lower left figure shows the gene expression of the activated stellate cell marker in the culture system, the central figure shows the image stained with aSWA after 5 days of activation, and the lower right figure shows the procollagen 1a secreted in the medium supernatant. The quantified results are shown. FIG. 12 shows a method for visualizing the activation of the activated hepatic stellate cell marker gene (ACTA2) along with the activation of quiescent hepatic stellate cells derived from iPS cells. In the figure, "RFP" indicates a gene encoding a red fluorescent protein, and "2A" indicates an intervening sequence encoding a peptide sequence of about 20 amino acid residues derived from virus. FIG. 13 shows the results of analyzing the expression of RFP accompanying activation by inducing differentiation of quiescent hepatic stellate cells from the ACTA2-RFP reporter iPS cells. FIG. 14 shows a method for evaluating hepatic stellate cell activation using reporter iPS cells obtained by introducing a reporter gene into quiescent hepatic stellate cells derived from iPS cells. FIG. 15 is a graph showing activation marker genes for agents (TGFb) that enhance activation of hepatic stellate cells and agents (A83-01, ICG-001) that suppress activation using the evaluation method shown in FIG. The result of having examined expression is shown. FIG. 16 shows the results of observing the fluorescence of the reporter protein (RFP) in the reporter iPS cells 5 days after the addition of each drug, using an FV3000 confocal laser scanning microscope. A is a fluorescence image of RFP, and B is a superimposition of images obtained by simultaneously staining the nuclei of cells using Hoechst 33342. FIG. 17 shows a screening system for therapeutic agents using an activation evaluation system for quiescent hepatic stellate cells derived from reporter iPS cells. The lower left diagram shows the results of automatically detecting the number of viable cells and RFP positive cells in each well (green: positive control, red: negative control). The lower right figure shows the validation results of the screening system. Green dots show the analysis result of each well and show the area of RFP fluorescence in hepatic stellate cells derived from ACTA2-RFP reporter iPS cells. Pink dots indicate the area of RFP fluorescence in hepatic stellate cells derived from normal iPS cells.

The present invention relates to a method for preparing quiescent hepatic stellate cells, which comprises the step of inducing differentiation of pluripotent stem cells and fractionating quiescent hepatic stellate cells using the NGFR-positive phenotype as an index. The present invention also relates to a method for introducing a reporter gene into the obtained resting hepatic stellate cells and screening a drug for treating a liver disease associated with liver fibrosis such as cirrhosis. The present invention is described in detail below.

1. Preparation of quiescent hepatic stellate cells derived from pluripotent stem cells (1) Pluripotent stem cells In the present specification, "pluripotent stem cells" are cells having self-renewal ability and pluripotency, which compose the living body. A cell having the ability to form any cell. "Self-renewal ability" refers to the ability to make the same undifferentiated cell as oneself from one cell. "Differentiation potential" refers to the ability of cells to differentiate. The pluripotent stem cells include, for example, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), mouse cells (multi-lineage differing cell sperm cells, and embryonic stem cells). (Germline stem cells: GS cells), embryonic germ cells (embryonic germ cells: EG cells) and the like, but are not limited thereto. The pluripotent stem cells used in the present invention are preferably iPS cells. The source of pluripotent stem cells may be any of mammals, birds, fish, reptiles and amphibians, and is not particularly limited. Mammals include primates (human, monkey, etc.), rodents (mouse, rat, guinea pig, etc.), cats, dogs, rabbits, sheep, pigs, cows, horses, donkeys, goats, ferrets, and the like. More preferably human.

In the present invention, the “iPS cell” refers to a cultured cell in which a somatic cell is initialized to an undifferentiated state by introducing a reprogramming factor into the somatic cell to impart pluripotency. As the reprogramming factor, for example, OCT3/4, KLF4, SOX2, and c-Myc can be used (Yu J, et al., Science, 2007; 318:1917-20), and c-Myc from the above four genes can be used. Except for 3 genes (TkDN4-M, 253G1 described later), OCT3/4 and SOX2 and LIN28 and Nanog can be used (Takahashi K, et al., Cell, 2007; 131:861-72). The form of introduction of these factors into cells is not particularly limited, and examples thereof include gene introduction using a plasmid, introduction of synthetic RNA, and direct introduction as a protein. Moreover, you may use the iPS cell produced by the method using microRNA, RNA, a low molecular compound, etc. In addition, iPS cells prepared from cells derived from healthy volunteers having an HLA (Human Leukocyte Antigen) type combination (HLA homozygote) that is considered to be less likely to cause rejection may be used. As the pluripotent stem cells including iPS cells, commercially available products or cells that have been distributed may be used, or newly prepared cells may be used. Examples of the iPS cells include 253G1 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, TkDN4-M. Strain, TkDA3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, hiPSC38-2 strain, MSC-iPSC1 strain, BJ-iPSC1 strain, etc. can do. Newly produced clinical grade iPS cells may be used. The origin of cells for producing iPS cells is not particularly limited, but, for example, fibroblasts or lymphocytes can be used.

(2) Hepatic stellate cell In the present invention, the term “hepatic stellate cell” typically refers to a stellate fibroblast-like cell having a vitamin A storage capacity, which is present in the liver and is in a dormant state (quiescent phase). Alternatively, it may be in an activated state. In the present specification, hepatic stellate cells in the dormant state (quiescent phase) are particularly referred to as “quiescent hepatic stellate cells” to distinguish them from activated hepatic stellate cells. Normally, even dormant hepatic stellate cells produce extracellular matrix such as collagen to form connective tissue, which is important for the normal organization of organ structures. On the other hand, when the hepatic stellate cells are activated, the hepatic stellate cells produce large amounts of excess collagen and inflammatory molecules.

The distinction between stationary hepatic stellate cells and activated hepatic stellate cells can be distinguished by measuring the protein production and gene expression mainly expressed in each hepatic stellate cell. When comparing the presence or absence of gene expression in each hepatic stellate cell, a method of measuring the mRNA expression using a marker gene specific to each gene can be used. Quantitative RT-PCR is a convenient method, although the amount of gene expression is not limited to quantification and analysis.

According to the present invention, as an index of gene expression of quiescent hepatic stellate cells derived from iPS cells, but not limited to, NGFR and/or the function of a gene expressed at the same time as NGFR (for example, LRAT, NES, HGF, And CYGB) can be used to sort stationary hepatic stellate cells. In the present specification, the above-mentioned protein or gene may be referred to as “quiescent marker”. "NGFR" is an abbreviation of N erve G rowth F actor R eceptor ( neurotrophic factor receptor), "LRAT" is retinol esterification enzyme L ecithin R etinol A cyl t ransferase ( lecithin - retinol acyltransferase ) stands for "NES" is nestin (a nestin), "HGF" is an abbreviation for H epatocyte G rowth F actor (hepatocyte growth factor), "CYGB" is Cytoglobin (site globin) Stands for. On the other hand, fibrosis-related gene markers that are expressed in cells when the quiescent hepatic stellate cells are activated include, but are not limited to, ACTA2, COL1A1 and the like (sometimes referred to as "activation marker" in the present specification. ) Can be used. “ACTA2” is α-smooth muscle actin 2, and “COL1A1” is an abbreviation for Collagen, Type I, Alpha 1 (type I collagen α1). Further, when iPS cells are used as pluripotent stem cells, the differentiation process can be observed using a gene specifically expressed in each stage as an index. For example, undifferentiated marker genes include OCT4 and NANOG, mesoderm marker genes include MESP1 and T, and lateral septal mesenchymal/hepatic stellate cell marker genes include HLX, LHX2, and FOXF1. .. When the expression levels of the marker genes of quiescent hepatic stellate cells and activated hepatic stellate cells are compared, the expression of "activation marker" in quiescent hepatic stellate cells is Is lower than the expression level of the marker, for example, 99% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, It is preferably 20% or less, 10% or less, 5% or less, and 0%. On the other hand, the expression of the "quiescent marker" in activated hepatic stellate cells is lower than the expression level of the marker in quiescent hepatic stellate cells, for example, 99% or less, 95% or less, 90% or less, It is preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 0%. In addition, the expression level of the activation marker of hepatic stellate cells after activation is preferably higher than the expression level of the markers of the same cells before activation, but not limited to 1.1 times, .2x, 1.3x, 1.5x, 2x, 3x, 4x, 5x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, It may be 80 times, 90 times, 100 times, 200 times, 300 times, 500 times, 1000 times or the like.

The primers that can be used in the quantitative RT-PCR are as follows.

(3) Method for Inducing Differentiation of Pluripotent Stem Cells into Quiescent Hepatic Stellate Cells The present invention is a method for inducing differentiation of pluripotent stem cells (eg, iPS cells) to prepare quiescent hepatic stellate cells, Provided is a preparation method including a step of three-dimensionally culturing pluripotent stem cells and/or a step of culturing at a low oxygen concentration. The present invention can induce quiescent hepatic stellate cell differentiation by three-dimensionally culturing pluripotent stem cells. Here, the term “three-dimensional culture” has the meaning commonly used by those skilled in the art, and can include culture forms such as spheroid culture, suspension culture, and gel culture. Further, the "gel culture" is a method of culturing cells in a gel substrate, and the gel substrate itself may or may not have an affinity for cells. May be. Further, differentiation of quiescent hepatic stellate cells can also be induced by culturing pluripotent stem cells at a low oxygen concentration together with or independently of the above three-dimensional culture. The low oxygen concentration and the culture period will be described later.

According to another aspect, the present inventors have previously reported a method of inducing hepatic stellate cell differentiation from iPS cells by using a Rock inhibitor (WO2016/148216; Koui, Y. ., et al., Stem. Cell. Rep., 2017, 9, 490-498). According to the present invention, a Rock inhibitor is used in the same manner as in the conventional method, but by changing the time of addition to the culture system, iPS cell-derived resting hepatic stellate cells that could not be obtained until now are obtained. I got it successfully.

The present invention provides a method for preparing quiescent hepatic stellate cells, which comprises the step of inducing differentiation of pluripotent stem cells (eg, iPS cells) and fractionating quiescent hepatic stellate cells using an NGFR-positive phenotype as an index. I will provide a. According to the present invention, a desired quiescent hepatic stellate cell can be obtained by adding a Rock inhibitor from day 4 after the initiation of differentiation induction of pluripotent stem cells. The known induction of hepatic stellate cell differentiation from iPS cells is to add a Rock inhibitor from the 6th day after the initiation of differentiation induction. The hepatic stellate cells obtained there are active when the expression of the marker gene is examined. It was a modified hepatic stellate cell. In Example 2 which will be described later, the hepatic stellate cells obtained by the present invention have a remarkable gene expression observed in quiescent hepatic stellate cells as compared with the hepatic stellate cells obtained by the conventional method. The gene expression seen in activated hepatic stellate cells cannot be observed. Similar results were also obtained by comparison with primary cultured hepatic stellate cells (Example 3). Thus, the start time of adding the Rock inhibitor is preferably 4 to 5 days before the 6th day from the start of differentiation induction.

The above-mentioned Rock inhibitor used for inducing differentiation of hepatic stellate cells from pluripotent stem cells refers to a drug that inhibits Rho-kinase (Rho-associated protein kinase). “Rho-kinase” is a serine-threonine protein kinase identified as a target protein of the low-molecular-weight GTP-binding protein Rho, and is known to be involved in various physiological functions such as smooth muscle contraction and cell morphological changes. Has been. The Rock inhibitor that can be used here is not particularly limited, and may be in the form of a low molecular compound, an antibody, an antisense compound, or the like. For example, Y27632 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide), Fasudil (hexahydro-1-(5-isoquinolinesulfonyl) -1H-1,4-diazepine), H-1152((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine ) And the like. Preferably, it is Y27632. The concentration at which the Rock inhibitor is added to the medium can be appropriately selected by those skilled in the art. For example, when Y27632 is used, the concentration added to the medium is 0.1 to 50 μM, preferably 1 to 30 μM, more preferably 5 to 20 μM, and even more preferably 10 μM.

As described above, it is preferable to start addition of the Rock inhibitor on the 4th day after the start of induction, but prior to that, the Rock inhibitor should be added on the first day (0th day) to the 1st day after the start of induction. It can be added appropriately. In this respect, it is no different from the conventional derivatization method. According to the present invention, as long as the desired quiescent hepatic stellate cells can be obtained by inducing differentiation from pluripotent stem cells, the type, concentration, and timing of addition of various additives other than the Rock inhibitor added to the culture system And the combination of additives are not limited. Such additives include, but are not limited to, BMP4 (factor that induces differentiation of pluripotent stem cells into mesoderm), ActA (activin A), bFGF, VEGF (vascular endothelial growth factor), SB431542 (TGF-β. Inhibitors), Dorsomorphin (BMP inhibitors) and the like. For BMP4, the time of addition to the medium is preferably immediately after the start of induction (day 0) to day 4, and the addition concentration is not limited, but is 0.5 to 50 ng/ml, 1 to 40 ng/ml, 2 to For example, 30 ng/ml is exemplified, and the addition concentration may be changed depending on the timing of addition. For example, the added concentration of BMP4 on the 1st to 4th days may be higher than the added concentration on the 0th to 1st days. Regarding ActA, the timing of addition to the medium is preferably 1 to 4 days after the start of induction, and the concentration of addition is not limited, but 1 to 20 ng/ml, 2 to 10 ng/ml, 3 to 5 ng/ml, etc. are exemplified. It Regarding ActA, the timing of addition to the medium is preferably 1 to 4 days after the start of induction, and the concentration of addition is not limited, but 1 to 20 ng/ml, 2 to 10 ng/ml, 3 to 5 ng/ml, etc. are exemplified. It Regarding bFGF, the timing of addition to the medium is preferably 1 to 4 days after the start of induction, and the concentration of addition is not limited, but examples thereof include 1 to 20 ng/ml, 2 to 10 ng/ml, and 3 to 5 ng/ml. It Regarding VEGF, the timing of addition to the medium is preferably 4 to 6 days after the start of induction, and the concentration of addition is not limited, but examples thereof include 1 to 30 ng/ml, 3 to 20 ng/ml, and 5 to 15 ng/ml. It For SB431542, the timing of addition to the medium is preferably on the 4th day after the start of induction, and the concentration of addition is not limited, but examples thereof include 1 to 15 μM, 2 to 10 μM, and 3 to 7 μM. For dorsomorphin, the time of addition to the medium is preferably on the 4th day after the start of induction, and the addition concentration is not limited, but may be 0.05 to 5 μM, 0.1 to 3 μM, 0.3 to 1 μM, etc. It The additive concentration of the above-mentioned additive may be any value within the range of the enumerated values, and, for example, in "1 to 20 ng/ml", 1, 2, 3, 4, 5, 1. 5, 3.5, etc. are included. In addition, in the process of inducing differentiation from pluripotent stem cells to obtain the desired resting hepatic stellate cells, the cell culture medium for culturing the cells can be appropriately selected by those skilled in the art. In an exemplary embodiment, the method of inducing differentiation of iPS cells to produce quiescent hepatic stellate cells is typically as shown in FIG. Specifically, using Stempro-34 SFM as the basic medium, 10 μM Y27632, 2 ng/ml BMP4 was added from day 0 to day 1 of induction, and 5 ng/ml ActA (activin 1 to day 4 from the day of induction was added. A) 5 ng/ml bFGF, 30 ng/ml BMP4 are added, and 10 ng/ml VEGF, 5.4 μM SB431542, 0.5 μM dorsomorphin, and 10 μM Y27632 are added 4 to 6 days after the start of induction. Do that. By culturing under these conditions until the 8th day, NGFR-positive cells can be collected to obtain quiescent hepatic stellate cells.

In general, oxygen culturing cells (O ₂₎ concentration is comparable to the O ₂ concentration in the atmosphere (approximately 20%), the induction method of the present invention, initiation of induction (day 0) - It is characterized by setting the O ₂ concentration to 4% until the 6th day. It may be difficult to keep the O ₂ concentration in the incubator constant at exactly 4% due to an artificial culturing operation, so that the O ₂ concentration may be increased for a very limited time. As described in Example 1 to be described later, the case where the O ₂ concentration was maintained at 4% and the culture was continued after the 6th day, and the case where the O ₂ concentration was increased to 20% and the culture was performed after the 6th day It was also found that culturing in an environment of high O ₂ concentration suppresses activation of hepatic stellate cells. Therefore, in the present invention, it can be said that in inducing differentiation into quiescent hepatic stellate cells, not only the above-mentioned addition timing of the Rock inhibitor but also increasing the O ₂ concentration in culture is an important factor.

Among pluripotent stem cells, iPS cells, ES cells, somatic stem cells and the like are suitable for suspension culture and form cell clusters with the progress of culture. Therefore, in culturing such cells, a low-adhesion incubator whose surface is coated with polyhydroxyethyl methacrylic acid (poly-HEMA), hydrogel, MPC (2-Methrylylethyl Phosphoryl Choline) polymer, collagen, etc. It can be carried out by suspension culture (spheroid formation) of cells on a non-adhesive incubator not coated with the cell adhesion molecule. The low-adhesion incubator and non-adhesive incubator may be commercially available or may be prepared in a laboratory. Furthermore, as a commercially available low-adhesive incubator, for example, EZSPHERE (a container for spheroid formation culture) (IWAKI), VECELL (registered trademark) plate (Bethel Co., Ltd.), NCP (NanoCulture Plate) (SCIVAX Life Sciences). Company), ULA (ultra-low adhesion surface) culture vessel (CORNING Co.), and commercially available non-adhesive incubators include, for example, petri dishes for suspension culture (manufactured by Nunc), and for suspension cell culture. Examples include commercially available petri dishes (Sumitomo Bakelite Co., Ltd.) and non-treatment plates (BD Falcon Co.). The seeding concentration of cells in various incubators can be adjusted appropriately. For example, when a 96-well type culture vessel is used, for example, 1×10 ³ to 1×10 ⁶ cells/well, preferably It may be seeded at 5×10 ³ to 5×10 ⁵ cells/well, more preferably 1×10 ⁴ to 1×10 ⁵ cells/well.

According to the present invention, when pluripotent stem cells (eg, iPS cells) are induced to differentiate to produce quiescent hepatic stellate cells, NGFR-positive cells are detected at the final stage of differentiation induction (eg, 8 days from the start of induction). Resting hepatic stellate cells can be sorted using the phenotype as an index. The process of collecting quiescent hepatic stellate cells using the NGFR-positive phenotype as an index is not particularly limited, but for example, fluorescence activated cell sorting (FACS) or magnetic cell sorting (MACS) ) Can be performed. Further, according to the present invention, the stationary phase hepatic stellate cells can be collected not only by the above-mentioned sorting but also by autofluorescence of cells or by centrifugation. For example, in the LRAT-positive period, cells emit autofluorescence due to vitamin uptake, and thus the cells can be fractionated using a means for detecting fluorescence and used as an index.

As described above, the quiescent hepatic stellate cells of the present invention are sorted by using the NGFR-positive phenotype as an index. For example, the cell fraction may be 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% with respect to all cells. As described above, 98% or more or 99% or more stationary phase hepatic stellate cells are included. In another aspect, the abundance ratio of quiescent hepatic stellate cells and activated hepatic stellate cells in the cell fraction obtained after the above-mentioned fractionation is higher than that of activated hepatic stellate cells. It is not particularly limited as long as there are many hepatic stellate cells, for example, 51:49, 55:45, 60:40, 70:30, 80:20, 90:10, 95:5, 97:3, 98:2. , 99:1, 100:0, etc.

The quiescent hepatic stellate cells obtained by the method of the present invention may be provided by being contained in a cell culture solution, a cell suspension, a cell preservation solution, or a hydrogel in a suitable container. Further, the quiescent hepatic stellate cells can be cryopreserved, for example, can be cryopreserved at −80° C. for 3 months or longer, 6 months or longer, 9 months or longer, or 12 months or longer. .. The stationary phase hepatic stellate cells of the present invention after freezing and thawing are not activated and can maintain stationary phase.

2. Preparation of quiescent hepatic stellate cells into which a reporter gene has been introduced A method for screening a drug for treating and preventing liver disease associated with liver cirrhosis and liver fibrosis caused by activation of hepatic stellate cells is provided. It According to the present invention, as described above, in activated hepatic stellate cells, since ACTA2 and COL1A1 can be detected as gene markers, the expression levels of these genes may be determined, or these genes themselves may be determined. Instead of determining the expression level of the gene, the expression level of the introduced reporter gene may be measured or its expression may be visualized. The hepatic stellate cells used in the screening method described below may be iPS cell-derived quiescent hepatic stellate cells into which a reporter gene functionally expressed in association with the expression of the above gene marker has been introduced.

Examples of reporter genes that can be introduced include, but are not limited to, fluorescent protein genes (red fluorescent protein, green fluorescent protein, yellow fluorescent protein, high-sensitivity green fluorescent protein, orange fluorescent protein, cyan fluorescent protein, etc.), luciferase gene (luc), Examples include beta-galactosidase gene (beta-gal), alkaline phosphatase gene (AP), secretory alkaline phosphatase gene (SEAP), beta-glucuronidase gene (GUS), and the like.

The reporter gene can be introduced into cells by genome editing techniques such as CRISPR/Cas9 system (Cong, L., et al., Science, 2013, 339, 819-823), TALEN (Transcribing Activator-Like Effector Nuclease). (Cermak, T., et al., Nucleic Acids Res., 2011, 39:e82), ZFN (Zinc Finger Nuclease) (Kim, Y.-G., et al., Proc. Natl. Acad. Sci. USA). , 1996, 93:1156-1160) and the like, which are known to those skilled in the art. Further, as a more general gene transfer method, a method of introducing a vector into which a reporter gene is incorporated may be used, and the gene transfer using the vector is not limited, but may be viral or non-viral gene transfer (for example, , Plasmid introduction, phage integrase, transposon, adenovirus, adeno-associated virus, and lentilvirus).

The combination of the gene of the gene marker and the reporter gene is not particularly limited, and may be, for example, ACTA2-RFP, ACTA2-lus, COL1A1-LacZ, or the like. In addition, an intervening sequence (for example, a gene encoding the 2A peptide sequence) may be appropriately introduced between the gene marker and the reporter gene. The purpose of introducing the intervening sequence varies. For example, the 2A peptide is a peptide sequence of about 20 amino acid residues derived from a virus, which is recognized by a protease (2A peptidase) which is internal to cells, and is cleaved at a position of one residue from the C terminus. Multiple genes linked to one unit by a 2A peptide (eg, ACTA2-RFP) can be transcribed and translated as a unit and then cleaved with a 2A peptidase.

Whether the hepatic stellate cells into which the reporter gene obtained by the method of the present invention has been introduced are quiescent hepatic stellate cells can be confirmed by examining the expression of each gene marker. In addition, the quiescent hepatic stellate cell into which the reporter gene of the present invention has been introduced can be provided as a cell fraction, like the quiescent hepatic stellate cell into which the reporter gene has not been introduced.

The quiescent hepatic stellate cells into which the reporter gene has been introduced, obtained by the method of the present invention, are provided by being contained in a cell culture medium, a cell suspension, a cell preservation solution, or a hydrogel in a suitable container. obtain. Furthermore, the quiescent hepatic stellate cells into which the reporter gene has been introduced can be cryopreserved, for example, at −80° C. for 3 months or more, 6 months or more, 9 months or more, or 12 months or more. It is possible to The quiescent hepatic stellate cells into which the reporter gene of the present invention has been introduced after freezing and thawing are not activated and can maintain the quiescent phase.

3. Evaluation Model and Screening Method for Effective Therapeutic Agents The culture system of quiescent hepatic stellate cells derived from pluripotent stem cells (particularly iPS cells) of the present invention is used for liver disease associated with hepatic stellate cell-induced liver cirrhosis and liver fibrosis. It serves as an evaluation model for drugs (test substances) that treat and prevent. More specifically, according to the present invention, a screening for selecting a test substance that suppresses activation of hepatic stellate cells as a candidate substance used as a drug for treating or preventing liver disease associated with fibrosis A method is provided, the screening method comprising:
(A) culturing quiescent hepatic stellate cells derived from pluripotent stem cells into which a reporter gene has been introduced;
(B) quiescent by adding a drug that activates quiescent hepatic stellate cells to the culture system of quiescent hepatic stellate cells, or collecting and reseeding the quiescent hepatic stellate cells and culturing in two dimensions Activating hepatic stellate cells to obtain activated hepatic stellate cells;
(C) adding a test substance to a culture system of activated hepatic stellate cells;
(D) comparing the expression level of the reporter gene before and after the addition of the test substance; and (e) for treating or preventing liver disease when the expression level of the reporter gene decreases after the addition of the test substance It may include selecting as a candidate substance for use in a drug.

In the present invention, the comparison of the expression level of the reporter gene depends on the type of the reporter gene used, and thus may be the comparison of the numerical values measured quantitatively, or the qualitative comparison of the observed images. May be According to the present invention, when the expression level of a reporter gene is decreased, it is selected as a test substance used in a drug for treating or preventing liver disease. The expression level is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80, 90%, or 100% from the expression level when hepatic stellate cells are activated. It is preferable.

As used herein, “treating” or “preventing” liver disease with a test substance can be used equivalently to the meaning of these general terms, eg, to reduce the effects of the disease. It has the meaning of controlling, abolishing, suppressing, relieving, reducing the severity of the disease, reducing the possibility of developing the disease, slowing the progression of the disease and/or curing the disease.

Drugs that can activate hepatic stellate cells in the screening method of the present invention include, but are not limited to, TGF-β, PDGF, chemokines, and the like. As described in Example 6 described later, in a culture system in which iPS cell-derived resting hepatic stellate cells were activated by TGF-β, A83-01 (TGF-β inhibitor) or Activation of hepatic stellate cells can be suppressed by adding ICG-001 (WNT inhibitor). The culture system to which the above drug is added may be a three-dimensional culture system, a gel culture system, or a two-dimensional culture system. Also, the terms "three-dimensional culture" and "gel culture" are as defined above, while the term "two-dimensional culture" has the same meaning as commonly used by one of ordinary skill in the art.

In another embodiment, the activation of quiescent hepatic stellate cells is performed not only by adding the above-mentioned activatable drug to the culture system, but also by inoculating the quiescent hepatic stellate cells in a two-dimensional culture system and culturing. It is possible. The expression level of the activation marker of hepatic stellate cells after being activated by two-dimensional culture is preferably higher than the expression level of the marker of the same cells before activation, but is not limited to 1.1 times. , 1.2 times, 1.3 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times It may be double, 80 times, 90 times, 100 times, 200 times, 300 times, 500 times, 1000 times or the like. The cell culture substrate used for the two-dimensional culture is not limited as long as it is a cell culture substrate capable of obtaining activated hepatic stellate cells. Such a cell culture substrate may be a plate or dish made of plastic such as polystyrene, polyethylene, polypropylene, polyethylene terephthalate, or the like, which has a hard bottom surface for cells, or glass. Further, the culture surface (bottom surface) of the above-mentioned cell culture substrate may be coated with a cell adhesion molecule such as collagen. The seeding concentration of cells in the two-dimensional culture can be adjusted appropriately.

4. Kit The present invention provides a kit suitable for screening a drug (test substance) for treating and preventing liver disease associated with cirrhosis and liver fibrosis. In one aspect, the kit of the present invention comprises a quiescent hepatic stellate cell (or a cell culture solution containing the cell) into which the reporter gene of the present invention has been introduced, a drug for activating the quiescent hepatic stellate cell ( (Above), a culture container, a cell culture medium, instructions for using the kit, and the like.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Quantitative RT-PCR
RNA was extracted using TRIzol reagent (Life Technologies) or NucleoSpin RNA XS (MACHEREY-NAGAL, Duren, Germany). The remaining genomic DNA was digested with DNaseI (Life Technologies 1oogies), and then single-stranded cDNA was synthesized using PrimeScript II 1st strand cDNA Synthesis Kit (Takara bio, Shiga, Japan). Quantitative RT-PCR was performed using SYBR Premix EX TaqII (Takara bio, Shiga, Japan), and data was calculated according to the ddCt method using β-actin as a standardized control.

Example 1: Induction of differentiation of iPS cells into quiescent hepatic stellate cells (1) Preparation of quiescent hepatic stellate cells Human iPS cells (TkDN4-M) (Tokyo University, Takayama, N., et al., J. Exp) Med., 2010, 207, 2817-2830) was cultured in a dish coated with vitronectin (Thermo Fisher Scientific). The iPS cells were detached from the dish using a StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific), and cultured at 1×10 ⁴ cells/cm ² in the Ultra-low attachment 6 wells, and seeded on the embryo-like plate. Using Stempro-34 SFM (Thermo Fisher Scientific) as a basic medium, 2 ng/ml BMP4, 10 μM Y27632 was added on the first day of induction 0 to 1 and 5 ng/ml ActivinA, 5 ng/ml bFGF, 30 ng on the first to fourth day of induction. /Ml BMP4 was added. After that, 10 ng/ml VEGF, 5.4 μM SB431542, 0.5 μM Dorsormorphin, and 10 μM Y27632 were added 4 to 8 days after the initiation of induction. It should be noted that 0 to 6 days after the start of induction was cultured in a 4% oxygen concentration environment, and 6 to 8 days after the start of induction was cultured in a 20% oxygen concentration environment. After induction for 8 days, quiescent hepatic stellate cells expressing NGFR were fractionated using a MoFLo XDP cell sorter (Beckman Coulter, Inc) (FIG. 1). Moreover, from the image of the cell morphology shown in FIG. 1, the process in which embryoid bodies were formed could be observed.

(2) Gene expression analysis during quiescent hepatic stellate cell differentiation process: Undifferentiated markers (OCT4, NANOG), mesoderm marker (0 days, 1, 4, 6 and 8 days after initiation of differentiation induction from iPS cells) Gene expression of MESP1, T), transseptal mesenchymal/astrocytic markers (HLX, LHX2, FOXF1) was analyzed by quantitative RT-PCR. Undifferentiated markers decreased with the progress of differentiation induction, mesoderm markers were highly expressed in the middle stage of differentiation induction, and lateral mesenchymal mesenchymal/astrocytic markers were highly expressed in the latter stages of differentiation induction (Fig. 2). .. From these results, it was found that the method of the present invention for inducing the differentiation of quiescent hepatic stellate cells from iPS cells is appropriate.

(3) Effect of Y27632 On the fourth day after the start of induction, 10 μM Y27632 was added to the culture system, and the effect of Y27632 to induce differentiation into quiescent hepatic stellate cells was analyzed by quantitative RT-PCR. For the hepatic stellate cell markers (NGFR, LRAT), the expression of each marker gene was induced by the addition of Y27632. On the other hand, the expression of activated hepatic stellate cell markers (ACTA2, COL1A1) was suppressed by the addition of Y27632 (FIG. 3). From these results, it was found that quiescent hepatic stellate cells can be induced by adding Y27632 on the fourth day after the start of induction.

(4) Examination of oxygen concentration Whether changes in the expression of activated hepatic stellate cell marker gene are observed under the condition of changing the oxygen concentration of 4% from 0 to 6 days after the start of induction to 6% from 6 days It was analyzed by quantitative RT-PCR. As shown in FIG. 4, the expression of activated stellate cell markers (ACTA2, COL1A1) was suppressed in a 20% oxygen concentration environment.

(5) Effect of Cell Sorting of NGFR-Positive Cells After induction of differentiation for 8 days, cells expressing NGFR (posi) and cells not expressing (nega) were collected using a MoFLo XDP cell sorter (Beckman Coulter, Inc), and before separation. The gene expression of the cells was compared by quantitative RT-PCR. Results are shown in FIG. In NGFR positive cells (posi), hepatic stellate cell markers (NGFR, LRAT, NES) were highly expressed, but activated hepatic stellate cell markers (ACTA2, COL1A1) were extremely low. On the other hand, NGFR negative cells (nega) had high expression of activated hepatic stellate cell markers (ACTA2, COL1A1). From this result, it was revealed that quiescent hepatic stellate cells can be distinguished from activated hepatic stellate cells by the presence or absence of NGFR expression, and NGFR can be an index for distinguishing both cells.

(6) Cell morphology of iPS cell-derived quiescent hepatic stellate cells The results of morphological observation of iPS cell-derived quiescent hepatic stellate cells on the 8th day from the start of induction are shown in FIG. As is clear from the image, the obtained cells showed a stellate fibroblast-like morphology like ordinary hepatic stellate cells.

Example 2: Comparison with hepatic stellate cells derived from iPS cells obtained by a known derivatization method Conventional hepatic stellate cells derived from iPS cells ("Ver. 1") (WO 2016/148216; Koui, Y) , Et al., Stem. Cell. Rep., 2017, 9, 490-498) and the iPS cell-derived hepatic stellate cells (“Ver. 2”) prepared in Example 1 were used for quantitative RT. -It was analyzed by PCR and compared. The hepatic stellate cell markers (NGFR, LRAT, NES) were highly expressed in the hepatic stellate cells prepared in Example 1, and the expression of the activated hepatic stellate cell markers (ACTA2, COL1A1) was extremely low. On the other hand, the hepatic stellate cells prepared by the conventional method had extremely high expression of the activated hepatic stellate cell marker (FIG. 7). From this, it was revealed that the hepatic stellate cells prepared by the conventional method are in the activated state and the hepatic stellate cells prepared in Example 1 are in the stationary phase.

Example 3: Comparison with primary culture hepatic stellate cells The gene expression in the iPS cell-derived hepatic stellate cells ("ver2") prepared in Example 1 and the gene expression of the primary culture hepatic stellate cells were compared by quantitative RT-PCR. investigated. Primary cultured hepatic stellate cells were obtained from Lonza. The results are similar to those in Example 2, but the hepatic stellate cell markers (NGFR, LRAT, NES) are highly expressed in the hepatic stellate cells prepared in Example 1, and the activated hepatic stellate cell markers (ACTA2, COL1A1) are The expression was extremely low (Fig. 8). On the other hand, in the primary cultured hepatic stellate cells, the expression of the hepatic stellate cell marker was extremely low, and the expression of the activated hepatic stellate cell marker was significantly high (FIG. 8). Similar to the results of Example 2, it was revealed that the hepatic stellate cells prepared in Example 1 were in the stationary phase and the primary cultured hepatic stellate cells were in the activated state.

Example 4: Vitamin A storage capacity The stationary phase hepatic stellate cells prepared in Example 1 were cultured, 10 µM retinol was added to the medium, and the cells were cultured for 4 days. After detaching the cells using 0.05% trypsin/0.5 mM EDTA, intracellular autofluorescence of vitamin A was detected by flow cytometry using CantoII (BD Biosciences). LX2 (human stellate cell line), primary hepatic stellate cells (2 lots (#1, #2)), and mesenchymal stem cells (MSC) were subjected to the same analysis for comparison. The results are shown in Fig. 9. Like the LX2 and primary cultured hepatic stellate cells, the quiescent hepatic stellate cells prepared in Example 1 stored vitamin A in the cells, and thus were found to function as hepatic stellate cells.

Example 5: Induction of activation of quiescent hepatic stellate cells The quiescent hepatic stellate cells prepared in Example 1 were seeded and cultured on a collagen-coated plastic plate. Gene expression was analyzed by quantitative RT-PCR before culturing and after 5 days of culturing. The expression of the activated stellate cell markers (ACTA2, COL1A1) was remarkably enhanced after the culture as compared with that before the culture (FIG. 10). It was found that the quiescent hepatic stellate cells prepared in Example 1 can induce activation in the culture system, like the quiescent hepatic stellate cells in the living body.

Additionally, the quiescent hepatic stellate cells prepared in Example 1 were induced to activate in a two-dimensional culture system (Fig. 11). The lower left diagram of FIG. 11 shows activated stellate cell markers (ACTA2, COL1A1, COL1A2) of hepatic stellate cells immediately after production (day 0) and after induction of activation (day 1, day 3, and day 5). , And COL3A1) gene expression analysis results. An increase in the activated stellate cell marker could be confirmed over time. The central figure shows an image in which the expression of the activated astrocyte marker aSMA (ACTA2) was confirmed by immunostaining. The lower right diagram is the result of measuring by ELISA the procollagen α1 (Pro-Collagen alpha1) secreted in the culture supernatant. From the above results, activation of quiescent hepatic stellate cells could be demonstrated at the gene level and the protein level.

Example 6: Validation of evaluation system based on activation of quiescent hepatic stellate cells (1) Preparation of reporter iPS cells Human iPS cells (TkDN4-M) were genetically modified using the CRISPR-Cas9 system. The stop codon of ACTA2, which is an activated hepatic stellate cell marker gene, was deleted, and RFP was inserted with the 2A peptide interposed (see FIGS. 12 and 13). Thus, quiescent hepatic stellate cells having the ACTA2-RFP reporter gene were obtained.

(2) Evaluation of hepatic stellate cell activation using reporter iPS cells Differentiated quiescent hepatic stellate cells were induced from iPS cells having the ACTA2-RFP reporter gene. The cells were seeded on a collagen-coated dish, and on the day after seeding, after confirming cell adhesion, the medium was replaced. At that time, a drug that enhances the activation of hepatic stellate cells (TGFb) and a drug that suppresses the activation (A83-01, ICG-001) were added to the medium (see FIG. 14). Gene expression was analyzed by quantitative RT-PCR before culturing and after 5 days of culturing (FIG. 15). The expression of the activated stellate cell markers (ACTA2, COL1A1) was remarkably enhanced after the culture as compared with that before the culture. The expression of these marker genes was enhanced by the addition of TGFb, and the expression was suppressed by the addition of A83-01 and ICG-001. The fluorescence of the reporter protein RFP in the cells after 5 days of culture was observed with an FV3000 confocal laser scanning microscope (OLYMPUS) (FIG. 16A). At the same time, Hoechst 33342 was used to stain the nuclei of cells (FIG. 16B). RFP fluorescence was suppressed under the conditions of addition of A83-01 and ICG-001 in correlation with gene expression.

Additionally, quiescent hepatic stellate cells were prepared from iPS cells having the ACTA2-RFP reporter gene, and then the expression of RFP was observed with a FV3000 confocal laser scanning microscope (OLYMPUS) over time in a two-dimensional culture system (Fig. 13 middle, lower left figure). No expression of RFP was observed in undifferentiated iPS cells or hepatic stellate cells immediately after activation induction (day 1). Further, when the activation was induced from the 3rd day to the 5th day, the fluorescence of RFP gradually became strongly observed.

Also, after inducing activation up to the 5th day, cells expressing RFP fluorescence were analyzed by flow cytometry analysis. No cells expressing RFP were observed in the quiescent stellate cells (day 0) immediately after preparation, but almost all the hepatic stellate cells after induction of activation (day 5) expressed RFP. It was understood (the lower right figure in FIG. 13). This revealed that undifferentiated iPS cells and quiescent hepatic stellate cells did not express RFP, but gradually expressed RFP upon activation, indicating that quiescent stellate cells derived from this ACTA2-RFP reporter iPS cell It was shown that the activation state of R. can be quantitatively evaluated using the expression of RFP as an index.

Example 7: Development of therapeutic drug screening system using activation evaluation system of quiescent hepatic stellate cells derived from reporter iPS cells In this example, the validity of the therapeutic drug screening system was verified (hereinafter referred to as an experiment). , Called "validation"). Quiescent hepatic stellate cells were generated from the ACTA2-RFP reporter iPS cells and then seeded in 384-well plates. Subsequently, Hoechst (nuclear stain) was added after 5 days of culture. An FV3000 confocal laser microscope was used to image the RFP fluorescence and Hoechst fluorescence for each well (lower left in FIG. 17). Next, using the image analysis software "cellSens" (OLYMPUS), based on the obtained image, the area of the RFP positive area (the activation degree of hepatic stellate cells) and the number of Hoechst dots (the number of cells) are calculated. It was calculated for each well (lower right in FIG. 17). Green dots in the lower right graph indicate the area of the RFP positive area in each well. The pink dots are the area of the RFP positive area when hepatic stellate cells derived from normal iPS cells were cultured by the same method (thus, the area is almost zero). The CV value shown in the graph indicates the degree of variation of the green dots, and the smaller the value, the less the variation is, which can be said to be a good screening system. Z'-factor is one of the indexes for evaluating the quality of the screening system, and is a value calculated from the dispersion of data and the intensity of measured values. Generally, it can be said that a Z′-factor of 0.5 or more is appropriate as a screening system.

The evaluation system using the iPS cell-derived quiescent hepatic stellate cells produced according to the present invention can be a useful tool for research and development of drug discovery such as therapeutic agents for fibrosis.

All publications and patent documents cited in this specification are incorporated herein by reference in their entirety. Although specific embodiments of the invention have been described herein for purposes of illustration, it will be understood by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Claims

A method for preparing quiescent hepatic stellate cells by inducing differentiation of pluripotent stem cells, comprising a step of three-dimensionally culturing pluripotent stem cells and/or a step of culturing at a low oxygen concentration.
The preparation method according to claim 1, wherein the pluripotent stem cells are cultured in the presence of about 4% O 2 from day 0 to day 6 of culture.
The preparation method according to claim 2, wherein the culture is performed at the same O 2 concentration as that in the atmosphere after the 6th day of culture.
The preparation method according to any one of claims 1 to 3, wherein the three-dimensional culture is spheroid culture, suspension culture, or gel culture.
The preparation method according to any one of claims 1 to 4, comprising a step of adding a Rock inhibitor.
The preparation method according to any one of claims 1 to 5, further comprising a step of incorporating a reporter gene into pluripotent stem cells before inducing differentiation.
The preparation method according to any one of claims 1 to 5, further comprising a step of inducing a reporter gene into a quiescent hepatic stellate cell during or after the induction of differentiation.
A quiescent hepatic stellate cell prepared by the method according to any one of claims 1 to 7.
A cell suspension containing the quiescent hepatic stellate cells according to claim 8.
A screening method for selecting a test substance that suppresses the activation of hepatic stellate cells as a candidate substance used as a drug for treating or preventing liver disease associated with fibrosis,
(A) culturing a quiescent hepatic stellate cell into which a reporter gene has been prepared, prepared by the method according to claim 6 or 7;
(B) by adding a drug that activates stationary phase hepatic stellate cells to the stationary phase hepatic stellate cell culture system, or recovering and reseeding the stationary phase hepatic stellate cells and performing two-dimensional culturing. Activating hepatic stellate cells to obtain activated hepatic stellate cells;
(C) adding a test substance to a culture system of activated hepatic stellate cells;
(D) comparing the expression level of the reporter gene before and after the addition of the test substance; and (e) for treating or preventing liver disease when the expression level of the reporter gene decreases after the addition of the test substance A screening method comprising selecting as a candidate substance used for a drug.
A kit for use in the screening method according to claim 10, which comprises the quiescent hepatic stellate cells according to claim 8 or the cell suspension according to claim 9.